Diethylstilbestrol (DES) is the first synthetic estrogenic compound prescribed to pregnant women to prevent miscarriage. Between 1947 and 1971, more than one million U.S. women were exposed to DES in utero, which predisposed them to reproductive tract patterning defects and clear-cell adenocarcinoma of the vagina or cervix at a young age. Changes in reproductive system development have subsequently led to infertility problems for these women. Even worse, DES was introduced into the environment for its ability to accelerate cattle growth. In 1971 alone, more than 27,600 kilograms of DES were used in livestock feed lots. Unfortunately, we still know very little about the molecular mechanism by which DES affects reproductive tract development. To address this mechanism is important because many synthetic and naturally occurring chemicals we are currently exposed to also mimic estrogen and could affect the health of the next generation in a similar fashion as DES did. This proposal will dissect the genetic pathways affected by DES during female reproductive tract (FRT) development. Our preliminary studies show that several developmental control genes are regulated by DES during critical period of uterine cytodifferentiation. In particular, homeodomain protein Msx2 appears crucial in counteracting the effect of DES on the devleoping FRT as DES induces very dramatic reproductive patterning defects in Msx2 mutants, resulted from altered molecular changes in these mutants. The present grant will continue to test the hypothesis that DES can change uterine epithelial cell fate by affecting genetic pathways governing uterine cytodifferentiation. In aim 1, the role of Msx2 in uterine and vaginal development and DES-induced FRT malformations will be rigorously examined. In aim II, we will use gain and loss of function approaches in vivo to examine whether Klf4 is both necessary and sufficient for DES-induced uterine metaplasia. Finally in aim III, we will test the hypothesis that DES affects luminal epithelial architecture through modulation of the Wnt pathway. By completing these studies, we should be able to build genetic pathways controlling uterine development and address how DES can cause abnormal FRT patterning through modulation of these pathways. Our long term goal is to use mouse as a model to study reproductive tract development and how exogenous factors can influence this process.